Caveolins are cholesterol binding proteins that can potentially regulate a variety of signal transduction pathways (Smart et al., (1999) Mol. Cell. Biol. 19, 7289-7304; Kurzchalia & Parton, (1999) Curr. Opin. Cell. Biol. 11, 424-431). For example, numerous researchers have demonstrated localization of proteins in caveolae, interaction of these proteins with caveolins, and the ability of overexpressed caveolins or peptides derived from caveolins to suppress or stimulate signaling functions in vitro or in cultured cells (Li et al., (1996) J. Biol. Chem. 271, 29182-29190; Razani et al., (1999) J. Biol. Chem. 274, 26353-26360; Nasu et al., (1998) Nat. Med. 4, 1062-1064; Garcia-Cardena et al., (1997) J. Biol. Chem. 272, 25437-25440). However, the importance of caveolins as modulators of signal transduction in vivo is controversial since caveolins-1 and -3, per se, are cholesterol binding proteins that deliver cholesterol from the endoplasmic reticulum to the plasmalemma thereby regulating signal transduction indirectly by modulating the cholesterol content of lipid raft domains and caveolae (Roy et al., (1999) Nat. Cell Biol. 1, 98-105; Sternberg et al., (1999) Nat. Cell Biol. 1, E35-37).
Recent studies have focused on the subcellular trafficking and regulation of endothelial nitric oxide synthase (eNOS). eNOS derived NO is necessary for the maintenance of systemic blood pressure, vascular remodeling, angiogenesis and wound healing (Huang et al., (1995) Nature 377, 239-242; Murohara et al., (1998) J. Clin. Invest. 101, 2567-0.2578; Rudic et al., (1998) J. Clin. Invest. 101, 731-736; Lee et al., (1999) Am. J. Physiol. 277, H1600-1608). eNOS is a dually acylated, peripheral membrane protein that targets to lipid raft domains and caveolae (Garcia-Cardena et al., (1996) Proc. Natl. Acad. Sci. USA 93, 6448-6453; Liu et al., (1997) J. Cell Biol. 137, 1525-1535). In caveolae, eNOS can physically interact with caveolins-1 and -3 by binding to their putative scaffolding domain located between amino acids 82-101 (Li et al., (1996) J. Biol. Chem. 271, 29182-29190) and this interaction, renders eNOS in its “less active” state (Garcia-Cardena et al., (1997) J. Biol. Chem. 272, 25437-25440; Ju et al., (1997) J. Biol. Chem. 272, 18522-18525; Michel et al., (1997) J. Biol. Chem. 272, 25907-25912). The data for this model was largely elucidated in vitro either using overexpression systems, fusion proteins or yeast-two hybrid screening to map the interacting domains.
In support of caveolin as a negative regulator of eNOS are studies showing that peptides derived from the scaffolding domain of caveolin-1 will disrupt the binding of eNOS to caveolin and dose-dependently inhibit NOS activity in vitro (IC50=1-3 μM) by slowing electron flux from the reductase to the oxygenase domain of NOS (Garcia-Cardena et al., (1997) J. Biol. Chem. 272, 25437-25440; Ju et al., (1997) J. Biol. Chem. 272, 18522-18525; Ghosh et al., (1998) J. Biol. Chem. 273, 22267-22271).
The present invention demonstrates that the treatment of one or more cells with a peptide comprising at least one caveolin scaffolding domain results in the reduction and/or elimination of one or more conditions or afflictions of the treated tissue, organ or organism. For example, treatment with a peptide comprising at least one caveolin scaffolding domain results in the reduction or elimination of inflammation and tumor cell angiogenesis and proliferation.
The present invention also demonstrates the use of antennapedia fusion peptides (“AP fusions”) to deliver bioactive peptides to cells of the vasculature in vitro and in vivo. Previous work utilizing this method of delivery has focused on the fusion of AP with oligonucleotides and small peptides for treatment of cells in culture. The uptake of AP bound cargo into cells is rapid, independent of membrane fluidity and is not affected by extremes in temperature (Derossi et al., (1996) J. Biol. Chem. 271, 18188-18193).
In the present experiments, Caveolin-1 scaffolding domain—antennapedia fusion peptides (“AP-Cav”) either in their biotinylated or rhodamine labeled forms, were internalized by the endothelium. Moreover, based on the anti-inflammatory actions of AP-Cav in vivo, the fusion peptides must be stable enough to survive first pass metabolism in the liver and pulmonary circuit to deliver the active peptides to the sites of inflammation. Preliminary evidence indicates that the AP-Cav peptides of the present invention do not increase blood pressure when delivered in vivo. Thus, the compositions and methods of the present invention provide useful approaches to delivering anti-sense oligonucleotides or as part of viral delivery systems for therapeutic cardiovascular gene targeting in vivo.
While not wishing to be bound by any particular theory, the fusion peptides of the present invention appear to be blocking one or more proteins that interact with or have the potential to interact with caveolin. Examples of such caveolin bound proteins include, but are not limited to, eNOS, the “Src Family” of tyrosine kinases (e.g., Src, Fyn, Lck, Yes, Lyn, Blk, Hick, Fgr, Yrk), Scr-like kinases, Ras proteins (e.g., Rho, Rac, Rab), Raf proteins (e.g., Raf-1, A-Raf, B-Raf), EGF receptors, and MAP kinases (e.g., Fus3) (Couet et al. (1997) J. Biol. Chem. 272, 6525-6533; Lewin (2000) Genes vii, Signal Transduction, pp. 801-834, Oxford University Press; Smith et al. (1997) Oxford Dictionary of Biochemistry & Molecular Biology, Oxford University Press).